Antioxidative activity of chitosans with varying molecular weights

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Abstract

Antioxidant activity of chitosans of different molecular weights (30, 90 and 120 kDa chitosan) in salmon (Salmo salar) was investigated. The progress of oxidation was monitored by employing the 2-thiobarbituric acid-reactive substances (TBARS) and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) scavenging assays. In general, all chitosans exhibited antioxidative activities in salmon. The addition of chitosans to salmon reduced lipid oxidation for seven days of storage. The TBARS values of salmon containing chitosan were significantly lower than those of the control (p < 0.01). At 0.2% (w/v) and 0.5% (w/v) concentrations, the TBARS with chitosan addition was decreased by 75% and 45%, respectively, over 15 days. At 1% concentration, the TBARS value with native chitosan addition was decreased by 32% after 15 days of storage. 90 kDa chitosan showed an increased DPPH free radical-scavenging activity with increasing concentration in the range of 0.2–1% (w/v). The free radical-scavenging activity of the 0.2 mM DPPH solution was saturated by 30 kDa chitosan at a concentration of ⩾0.7% (w/v), resulting in a strong antioxidant activity of approximately 85%. This was comparable to the DPPH free radical-scavenging activity of BHT.

Introduction

Chitosan (poly-β-1, 4-linked glucosamine) is a cationic polysaccharide made from alkaline N-deacetylation of chitin. It has attracted attention as a biomedical material, owing to its unique biological activities which include antitumor, immuno-enhancing effects and antibacterial activity in combination with its low toxicity (Kim et al., 2003, Suzuki et al., 1986, Suzuki, Tokoro, et al., 1986). It can be used for the chelation of transition metals (Muzzarelli, Muzzarelli, & Terbojerich, 1997), edible coatings for fruit and vegetables (Castellanos-Perez et al., 1988, El-Ghaouth et al., 1992, Park, 1999) packaging films (Butler et al., 1996, Caner et al., 1998, Park et al., 1999, Rhim et al., 1998) and waste water purification (Knorr, 1991). The application of this polysaccharide in the food industry and medicine is, however, limited because of its high molecular weight (MW) resulting in its low solubility in aqueous media (Ilyina, Tikhonov, Albulov, & Varlamov, 2000). It is important to improve the water solubility of chitosan so that it can be incorporated into different films which will expand its usefulness in the food industry.

Chitosan hydrolysate can be prepared by enzyme hydrolysis (Amano and Ito, 1978, Hirano and Nagano, 1989, Sashiwa et al., 1989) or acid hydrolysis (Horowitz et al., 1957, Hwang et al., 2002). The enzymatic method has the advantage over chemical reactions due to the fact that it takes place under mild conditions and does not create environmental problems (Mekkriengkrai, Chirachanchai, & Pichyangkura, 2001). It also has the minimum effect on the chemical nature of the reaction product. There are several reports of different enzymes such as lipase, lysozyme, chitinase and chitosanase being utilized in this procedure (Aiba, 1994, Ilyina et al., 2000, Shin et al., 2001, Stoyachenko et al., 1994).

Recently, the antioxidant activity of chitosan and its derivatives has attracted attention. Xie, Xu, and Liu (2001) studied the antioxidant activities of water-soluble chitosan derivatives which were considered to be hydroxyl radical scavengers. Antioxidant activities of different MW chitosans in salmon may be attributed to their metal-bonding capacities. Several sources of protein-bound iron exist in fish tissue, e.g., myoglobin, hemoglobin, ferritin and transferrin. The iron bound to these proteins may be released during storage thus activating oxygen and initiating lipid oxidation (St. Angelo, 1996). Xue, Yu, Hirata, Terao, and Lin (1998) reported that water-soluble chitosans may chelate metals or combine with lipids resulting in a significant antioxidative effect. Peng et al., 1998, Winterowd and Sandford, 1995 showed that chitosans retard lipid oxidation by chelating ferrous ions present in the system, thus eliminating their prooxidant activity or their conversion to ferric ion. Furthermore, amino groups in chitosans may participate in the chelation of metal ions. Kamil, Jeon, and Shahidi (2002) demonstrated that among the different viscosity chitosans, 14 cp chitosan was more effective than the higher viscosity chitosans in preventing lipid oxidation in the herring flesh model system. Darmadji and Izumimoto (1994) observed the effectiveness of chitosan treatment on the inhibition of lipid oxidation of beef. The addition of 1% chitosan resulted in a 70% reduction of thiobarbituric acid-reactive substances (TBARS) values in meat after 3 days of storage at 4 °C.

The highly unsaturated fatty acids commonly found in seafoods are particularly sensitive to oxidative change during storage (Hsieh and Kinsella, 1989a, Shahidi, 1997). It has been proposed that lipid oxidation in fish may be initiated and promoted by a number of mechanisms including autoxidation, photosensitized oxidation, lipoxygenase, peroxidase, and microsomal enzymes (Hsieh & Kinsella, 1989).

The heavy metal–polymer complexes are considered to form as a result of dative bonding with chitosan (Kamil et al., 2002). This involves the donation of nonbonding pairs of electrons from the nitrogen, and/or the oxygen of the hydroxyl groups, to a heavy metal ion (Tual, Espuche, Escoubes, & Domard, 2000).

Synthetic antioxidants and chelating agents can be added to food products to prevent lipid oxidation (Kamil et al., 2002). However, the growing consumer demand for food devoid of synthetic antioxidants has focused research on the development of new natural preservatives (Matsugo et al., 1998). Several sources of natural antioxidants are known (Shahidi, 1997), and some of them, such as those of rosemary and sage, are currently used in a variety of food products. Fundamental studies on chitosan as a natural antioxidative agent in fish have not been conducted (Kamil et al., 2002). The objective of this study was to examine the effect of chitosans of different molecular weights as antioxidative agents in salmon based on the measurement of 2-thiobarbituric acid-reactive substances (TBARS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity.

Section snippets

Materials

All chemicals used in this study were obtained from commercial sources and were of the highest purity available. 2-Thiobarbituric acid (TBA) was purchased from Sigma Chemical Co. (St. Louis, MO). Native chitosan (MW 120 kDa, 84.71% deacetylation) and 40 cp (MW 80 kDa, 80.77% deacetylation) chitosan were purchased from Kimitsu Co. (Tokyo, Japan). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical was obtained from Sigma–Aldrich Co. (St. Louis, MO).

Preparation of degraded chitosan

Degraded chitosan was prepared enzymatically from native

IR spectra analyses

Infrared spectroscopy has been used to determine the structure of chitin and chitosan (Kurita, 1986). Fig. 1 shows the FTIR spectra of degraded chitosan and native chitosan. The spectral patterns of degraded and native chitosan used in this study were similar to that of chitosan reported by Lim, Khor, and Koo (1998). The main characteristic peaks of chitosan were at 3455 (O–H stretch), 2867 (C–H stretch), 1598 (N–H bend), 1154 (bridge O stretch), and 1094 cm−1 (C–O stretch). In the spectrum of

Conclusions

Incorporation of 0.2%, 0.5% and 1% chitosan with various molecular weights into salmon resulted in reduced lipid oxidation. The 30 kDa chitosan which has high water solubility used in this study may be considered as a potential natural antioxidant for stabilizing lipid containing foods to prolong shelf life as well as being an excellent antimicrobial agent. The present study should provide a possible application of chitosan as a food additive to high lipid food systems.

Acknowledgments

The authors thank Dr. Valerie A Paynter for assistance in preparation of the manuscript and Dr. Hill for the statistical analysis.

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    This research was carried out in the Department of Packaging Science, Clemson University, Clemson, SC 29634-0370, USA.

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